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J Vis Exp. 2016 Feb 27;(108):53563. doi: 10.3791/53563.

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates.

Author information

1
Laboratory for Molecular Cancer Biology, Department of Radiation Oncology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University.
2
Department of Radiation Oncology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University.
3
Laboratory for Molecular Cancer Biology, Department of Radiation Oncology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University; athanasios.vasilopoulos@northwestern.edu.

Abstract

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

PMID:
26966987
PMCID:
PMC4828201
DOI:
10.3791/53563
[Indexed for MEDLINE]
Free PMC Article

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